Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-06-05 - 2001-06-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Isophorone diamine of Degussa AG, batch no. 050301. Purity 99.8 %; impurity water 0.01 %

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
- Source: Charles River Laboratories, L'Arbresle (France)
- Age: 10-11 weeks
- Weight at study initiation: 206-301, mean 245 g. Mean weights in the four groups were similar.
- Housing: single
- Diet: Pelleted maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
- Vehicle: Water purified by reverse osmosis
- Concentration in vehicle: 1, 5, or 25 g/l
- Total volume applied: 10 ml/kg bw/treatment
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analyses demonstrated satisfactory stability and agreement between nominal and actual concentrations of the test material.
Details on mating procedure:
Females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as day 0 post-coitum.
Duration of treatment / exposure:
day 6 to day 19 post-coitum inclusive
Frequency of treatment:
daily
Duration of test:
until day 20 post-coitum
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 50, or 250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
24 (females only); only the first 20 pregnant females were taken into consideration for fetal examinations
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were determined based on the results of a preliminary study

Examinations

Maternal examinations:
PARAMETERS ASSESSED DURING STUDY:
- Mortality: daily (twice during treatment period)
- Clinical signs: daily (twice during treatment period)
- Body weight gain: Days 2, 6, 9, 12, 15, 18, 20
- Food consumption: Cumulative for days 2-6; 6-9; 9-12; 12-15; 15-18; 18-20
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): Principal thoracic and abdominal organs, gross evaluation of placentas
Ovaries and uterine content:
- Examination of uterine content: Weight of gravid uterus, number or corpora lutea, number and distrubution of implantation sites (or uterine scars), number and distrubution of early and late resorptions, number and distribution of dead and live fetuses
Fetal examinations:
- Examination of fetuses: weight, sex, detailed external examination (all); soft tissue including all organs and structures of head, neck, thorax, and
abdomen (one half); skeleton including bone structures and cartilage of head, spine, rib cage, pelvis, limbs (other half)
Statistics:
- Group mean values +/- standard deviation (one-way analysis of variance and Dunnett test): Maternal body weight and food consumption, fetal body weight and number of corpora lutea, implantations, fetuses and resorptions
- Proportions (Fisher exact probability test): Pre-implantation loss, post-implantation loss, fetal findings

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality and day of death: One female of the high-dose group was found dead on day 16. This death was attributed to an effect of hold-up in the
esophagus following gavage. No other mortalities were observed.
- Clinical signs: The only treatment-related clinical sign was ptyalism in most females of the 250 mg/kg bw/day group from day 11, 12, 13, or 14 until hysterectomy. Loud breathing and presence of material in the mouth, probably due to hold-up in the esophagus, were recorded in 4 females. These observations might be the consequence of the corrosive properties of the test item (pH between 10 and 12).
- Number pregnant per dose level:
Control 24; low- and high-dose 23; mid-dose 22
- Number aborting: No abortion in any group
- Number of resorptions: No total resorption in any group
- Pre and post implantation loss: No treatment related findings were observed at any dose level.
- Body weight gain: Not affected in low and mid dose groups. A significant decrease (-35 %) was observed in the high-dose group after the first three days of treatment. Thereafter, the body weight was similar to that of the controls. The net body weight gain was also significantly lower (-25 %) at
this dose level.
- Food/water consumption: Not affected in low and mid dose groups; a slightly significant decrease was observed in the high dose group during the
treatment period.
- Gross pathology incidence and severity: No treatment related findings were observed at any dose level. An exception are whitish foci on the lung in
the decedent female.

Effect levels (maternal animals)

Dose descriptor:
NOEL
Effect level:
50 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- Litter size and weights: No treatment related findings were observed at any dose level.
- Sex ratio: No treatment related findings were observed at any dose level.
- External abnormalities: No treatment related external malformations or variations were observed at any dose level.
- Soft tissue abnormalities: No treatment related soft tissue malformations or variations were observed at any dose level.
- Skeletal abnormalities: No statistically significant treatment related skeletal malformations or variations were observed at any dose level.
There was a statistically insignificant increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group
(106/134 fetuses = 79.1%, p<0.01 were affected vs. 88/130 = 67.7% in control group).
In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the
rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5% in control group.
When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight
fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
> 250 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

 No teratogenic or embryofetotoxic effects were recorded at any dose level.

Applicant's summary and conclusion

Conclusions:
Isophorone diamine did not show any teratogenic or embryofetotoxic effects in a gavage study with rats performed in accordance with OECD TG 414 (2001) up to and including the highest tested dose level of 250 mg/kg bw/day. The NOEL for maternal toxicity was 50 mg/kg bw/day, effects at 250 mg/kg bw/day were reduced food consumption and reduced body weight gain. The NOAEL for developmental toxicity is > 250 mg/kg bw/day
Executive summary:

Based on the results of a dose finding study, three groups of 24 mated female Sprague-Dawley rats received 3-aminomethyl-3,5,5-trimethylcyclohexylamine by daily oral administration (gavage) at 0 (water = control), 10, 50 and 250 mg/kg/day from day 6 to day 19 post-coitum inclusive. On day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The study was designed according to OECD TG 414.There was no treatment-related death in any of the dams. Clinical signs were not observed, except for ptyalism in most females of the 250 mg/kg/day group (from day 11, 12, 13 or 14 post-coitum until hysterectomy; effect not considered as adverse). Loud breathing and hold-up in the esophagus were recorded in 4 females of this group and a significantly lower body weight gain (-35%) was recorded after the first three days of treatment. Thereafter, the body weights were similar to that of the controls. Over the whole treatment period, the difference remained slight (-10 %, not statistically significant). The net body weight gain was also significantly lower at this dose-level (-25 %) when compared to the control group. In the 250 mg/kg/day group, a significant decrease in food consumption was recorded during the treatment period (-7%), with a more marked effect during the first three days of treatment (-21 %).Abortions or total resorptions were not observed in any of the groups, nor were there any macroscopic findings that were ascribed to treatment with the test item. No treatment related effects were observed on pre- or post-implantation loss, fetal weight or sex-ratio. With respect to the fetuses, no test item related external, soft tissue or skeletal malformations or variations were detected. There was an increase in fetal incidence of incomplete ossification of the 5th sternebra in the 250 mg/kg bw/day group (106/134 fetuses = 79.1 %, p < 0.01 were affected vs. 88/130 = 67.7 % in control

group, statistically insignificant on a fetus/litter basis). Because these findings are of low concern and occur only in the presence of maternal toxicity, they are considered to be secondary. In the same group there was a statistically nonsignificant increase in fetal incidence of incomplete ossification of the rib(s) (9/137 fetuses = 6.7 % vs. 2/130 = 1.5 % in control group. When ossification was incomplete, cartilage was generally present, demonstrating that the skeletal variations recorded corresponded to slight fluctuations in the time of ossification rather than being a persistent alteration. In conclusion, these findings were considered to be incidental and of no toxicological significance. The NOEL for maternotoxicity was 50 mg/kg/day and the NOAEL for embryonic development was >250 mg/kg/day.